Early induction of cytokine release syndrome by rapidly generated CAR T cells in preclinical models

Cytokine release syndrome (CRS) is a significant side-effect of conventional chimeric antigen receptor (CAR) T-cell therapy. To facilitate patient accessibility, short-term (st) CAR T cells, which are administered to patients only 24 h after vector exposure, are in focus of current investigations. Their impact on the incidence and severity of CRS has been poorly explored. Here, we evaluated CD19-specific stCAR T cells in preclinical models. In co-culture with tumor cells and monocytes, stCAR T cells exhibited anti-tumoral activity and potent release of CRS-related cytokines (IL-6, IFN-γ, TNF-α, GM-CSF, IL-2, IL-10). When administered to NSG-SGM3 mice, stCAR T cells, but not conventional CAR T cells, induced severe acute adverse events within 24 h, including hypothermia and weight loss, as well as high body scores, independent of the presence of tumor target cells. Human (IFN-γ, TNF-α, IL-2, IL-10) and murine (MCP-1, IL-6, G-CSF) cytokines, typical for severe CRS, were systemically elevated. Our data highlight potential safety risks of rapidly manufactured CAR T cells and suggest NSG-SGM3 mice as sensitive model for their preclinical safety evaluation.

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EMBO Molecular Medicine has a "scooping protection" policy, whereby similar findings that are published by others during review or revision are not a criterion for rejection.Should you decide to submit a revised version, I do ask that you get in touch after three months if you have not completed it, to update us on the status.I look forward to receiving your revised manuscript.This is a very interesting paper exploring the murine models for assessing CRS in CAR T treatment and their data provide important clues.However, the controls used in the experiments were not ideal.This paper could improve significantly if this caveat is solved.
Referee #1 (Remarks for Author): The authors assessed the risk of cytokine release syndrome (CRS) from short-term (st) CAR T cells in a murine model.Their data recapitulated the early onset of CRS in the murine model and emphasized the importance of assessing the stCAR T cells used in clinical trials.Compared with standard CAR T cells, stCAR T cells showed a similar stable expression level of CARs day seven posttransduction and elicited comparable killing of target cells in vitro.However, stCAR T cells had a higher percentage of naïve T cells with a less differentiated phenotype.Infused into mice bearing NALM6 tumor, stCAR T cells triggered typical CRS-related symptoms in mice, which was probably exaggerated by monocytes.It was also very interesting that in this murine model, stCAR T cells could trigger the CRS symptoms in the absence of target tumor cells.These findings are very interesting as there are few accepted murine models to assess the CRS, especially regarding stCAR T cells.Their data could provide helpful information for developing short-term CAR T therapies.I recommended publishing this paper with minor revisions.
Minor revisions: 1.It is very interesting that stCAR T cells alone could trigger CRS symptoms in mice without recognizing NALM6 target cells.As the authors discussed, "the high amounts of VSV-G protein on stCAR T cells trigger the innate immune response similarly to a viral infection."Therefore, it is critical to make a well-controlled experiment to elucidate this question.In Figure 2 and S2, the ideal control for the mice experiment should be mock transduced T cells using the same virus backbone in addition to untransduced activated T cells.This control could answer this fundamental question.2. Similarly, in Figure 3, it might be possible that stCAR T cells could release CRS-related cytokines in vitro without target cells.Therefore, the ideal controls should include mock transduced T cells and effector T cells alone.The expected experiment groups should be: 1) stCAR T cell no Monocytes no NALM6, 2) stCAR T cells no Monocytes + NALM6, 3) stCAR T cells + Monocytes no NALM6, 4) stCAR T cells + Monocytes + NALM6; 5) Mock T cell no Monocytes no NALM6, 6) Mock T cells no Monocytes + NALM6, 7) Mock T cells + Monocytes no NALM6, 8) Mock T cells + Monocytes + NALM6.3. The authors thought stCAR T cells migrate more to the bone marrow, which caused the low number of stCAR T cells in peripheral blood.It would be wonderful if the authors could run a flow with stCAR and CAR T cells using a panel of chemokine receptors and integrins.Certain chemokine receptors, such as CXCR4, have a significant role in cell migration to bone marrow.4. In Figure S2, D and E showed the human T cells and NALM6 cell in the bone marrow.I thought the two groups were mice treated with T cells and stCAR T cells.Why was there only one histogram?5.In Figure S9, E, there were much fewer stCAR T cells than CAR T cells in bone marrow.Could the authors discuss this regarding the data in Figure 3? 6.The calculation equation for cytotoxicity was not correctly written.It should be: % Specific lysis = 100 X (1 -(% viable target cells in the presence of CAR T cells)/(% viable target cells in the presence of untransduced T cells)) Referee #2 (Comments on Novelty/Model System for Author): CAR-T therapy is effective but very costly and stressful for the patient.There is significant toxicity, and there is shortage of capacity.Shorter manufacture is a possible solution, but has risks which this animal study documents carefully Referee #2 (Remarks for Author): This animal model revealing greater toxicity due to greater cytokine levels might be used to optimise the therapy of CRS, and its prevention by optimising anticytokine therapy.This is too difficult to do in humans, but might comparing therapies to CRS in these mice be useful for patients?AntiIL6 receptor is standard of care but its never been compared to antiTNF or antiIFNg or ant combinations.

Referee #3 (Remarks for Author):
In the manuscript entitled "Early induction of cytokine release syndrome by rapidly generated CAR T cells in a preclinical mouse model", Ho et.al. seek to observe the safety of short-term expanded CAR T (st) focusing specifically on residual vector particle expression and induction of cytokine release syndrome (CRS).Given the push of the field to use shorter expansion times for cell products, this study offers important insight for the advancement of CAR T cells in the clinic.While the study is fairly easy to follow and has great potential, there are some concerns with the presented work.
Major revisions: 1. How do CAR T compare to stCAR T in the in vitro model with Nalm6 and monocytes?Are there differences in the cytokine expression or do these look similar to the in vivo model?Given that the NSG-SGM3 strain expresses human GM-CSF, it could be hiding potential differences of this cytokine between groups.This could be explored using the in vitro model.T cells alone compared to stCAR T are not necessarily helpful to compare.
2. The authors show that CAR T are twice as prevalent in the bone marrow of non-tumor-bearing mice compared to stCAR T cells.How does their expression compare in the presence of Nalm6 tumor in NSG-SGM3 mice?This would be more relevant to treatment of cancer-bearing patients.
3. How does the murine innate cell compartment (as shown in Fig. 2F) differ between Nalm6-bearing mice treated with CAR T versus stCAR T?
Minor revisions: 1. Fig. 1C: Almost 80% of CAR T cells are double negative (DN)--is this data correct?I would expect more CAR+ by this point in culture.
2. Labeling is off in the text for Fig. 1E-I.
3. Data is not presented in order--i.e.some of Suppl.Fig. 3 is discussed in the text prior to jumping back to Supp.Fig. 2.This made it difficult to follow and would flow better if the data was introduced in the correct order.4. In Supp.Fig. 8, changing the axis of the middle plot (CAR T) to a max of 8 to match the other two graphs will make your data more striking.As it's currently graphed, it appears that the severity of CRS is comparable between stCAR T and CAR T.
5. Interferon gamma is written as both IFN-g and INF-g in the manuscript--please choose one and make consistent.This is a very interesting paper exploring the murine models for assessing CRS in CAR T treatment and their data provide important clues.However, the controls used in the experiments were not ideal.This paper could improve significantly if this caveat is solved.
Thank you very much for this positive feedback.We have added further data and controls as described below.
Referee #1 (Remarks for Author): The authors assessed the risk of cytokine release syndrome (CRS) from short-term (st) CAR T cells in a murine model.Their data recapitulated the early onset of CRS in the murine model and emphasized the importance of assessing the stCAR T cells used in clinical trials.Compared with standard CAR T cells, stCAR T cells showed a similar stable expression level of CARs day seven post-transduction and elicited comparable killing of target cells in vitro.However, stCAR T cells had a higher percentage of naïve T cells with a less differentiated phenotype.Infused into mice bearing NALM6 tumor, stCAR T cells triggered typical CRSrelated symptoms in mice, which was probably exaggerated by monocytes.It was also very interesting that in this murine model, stCAR T cells could trigger the CRS symptoms in the absence of target tumor cells.These findings are very interesting as there are few accepted murine models to assess the CRS, especially regarding stCAR T cells.Their data could provide helpful information for developing short-term CAR T therapies.I recommended publishing this paper with minor revisions.
Thank you for this positive feedback, much appreciated.
Minor revisions: 1.It is very interesting that stCAR T cells alone could trigger CRS symptoms in mice without recognizing NALM6 target cells.As the authors discussed, "the high amounts of VSV-G protein on stCAR T cells trigger the innate immune response similarly to a viral infection."Therefore, it is critical to make a well-controlled experiment to elucidate this question.In Figure 2 and S2, the ideal control for the mice experiment should be mock transduced T cells using the same virus backbone in addition to untransduced activated T cells.This control could answer this fundamental question.
We totally agree, that your proposed experimental setting will be a valuable control, however, it will not be possible to perform this additional animal experiment in due course.We therefore decided to focus on the ex vivo experiment you suggested below, which confirmed that the presence of CAR plus the VSV glycoprotein were causative for the cytokine release we observed.The data were added as additional main figure (Fig. 6).3, it might be possible that stCAR T cells could release CRS-related cytokines in vitro without target cells.Therefore, the ideal controls should include mock transduced T cells and effector T cells alone.The expected experiment groups should be: Thank you for suggesting this experimental setup, which we followed with slight adaptations.The obtained data were clear-cut in showing that release of CRS-related cytokines does not occur when the vector particles delivered GFP or were empty (VLP).In contrast, massive cytokine release was determined with stCAR T cells, which further increased in presence of monocytes.The presence of tumor cells made some difference for the secretion of particular cytokines.The data are now shown in an additional main figure (Fig. 6) and mentioned in the Discussion (line 292 and following).

Similarly, in Figure
3. The authors thought stCAR T cells migrate more to the bone marrow, which caused the low number of stCAR T cells in peripheral blood.It would be wonderful if the authors could run a flow with stCAR and CAR T cells using a panel of chemokine receptors and integrins.Certain chemokine receptors, such as CXCR4, have a significant role in cell migration to bone marrow.
We are sorry if we were not precise enough in our statement on the bone marrow migration.In fact, there were more CAR T cells in bone marrow after administration of conventional than of stCAR T cells.We have now added a data set quantifying CAR T cells in bone marrow of the mouse study shown in Fig. 4. Also here, there were about twice as many conventional CAR T cells in BM than stCAR T cells (Fig. EV3).The misleading statement has been rephrased (line 191-195).
4. In Figure S2, D and E showed the human T cells and NALM6 cell in the bone marrow.I thought the two groups were mice treated with T cells and stCAR T cells.Why was there only one histogram?
This shows the cells migrated to BM for the stCAR T group only, since these mice had to be sacrificed on day one after cell administration whereas the group having received T cells was sacrificed after 12 days.Therefore, we have do not have values for the amounts of T cells on day one.S9, E, there were much fewer stCAR T cells than CAR T cells in bone marrow.Could the authors discuss this regarding the data in Figure 3?

In Figure
We are sorry for explaining this improperly.As mentioned above, we have now added the comparison stCART and conventional CART in BM for the mouse study in Fig. 4 (Fig. EV3A).The comparison related to the mouse study in Fig. 5 is now shown in Fig. EV5 (previously Fig. S9).Both data sets are in full agreement showing about half as many stCART in BM than conventional CART.We are now better explaining this (line 191-195).
6.The calculation equation for cytotoxicity was not correctly written.It should be: % Specific lysis = 100 X (1 -(% viable target cells in the presence of CAR T cells)/(% viable target cells in the presence of untransduced T cells)) Thank you for pointing this out.The formula has been corrected.

Referee #2 (Remarks for Author):
This animal model revealing greater toxicity due to greater cytokine levels might be used to optimise the therapy of CRS, and its prevention by optimising anticytokine therapy.This is too difficult to do in humans, but might comparing therapies to CRS in these mice be useful for patients?AntiIL6 receptor is standard of care but its never been compared to antiTNF or antiIFNg or ant combinations.
Thank you very much for the positive feedback and the suggestion on using the mouse model.We do now mention this in the final sentence of the Discussion.

Referee #3 (Remarks for Author):
In the manuscript entitled "Early induction of cytokine release syndrome by rapidly generated CAR T cells in a preclinical mouse model", Ho et.al. seek to observe the safety of short-term expanded CAR T (st) focusing specifically on residual vector particle expression and induction of cytokine release syndrome (CRS).Given the push of the field to use shorter expansion times for cell products, this study offers important insight for the advancement of CAR T cells in the clinic.While the study is fairly easy to follow and has great potential, there are some concerns with the presented work.
Major revisions: 1.How do CAR T compare to stCAR T in the in vitro model with Nalm6 and monocytes?Are there differences in the cytokine expression or do these look similar to the in vivo model?Given that the NSG-SGM3 strain expresses human GM-CSF, it could be hiding potential differences of this cytokine between groups.This could be explored using the in vitro model.T cells alone compared to stCAR T are not necessarily helpful to compare.
Thank you for your positive and constructive feedback.In the revised manuscript we have added data of an additional in vitro experiment designed according to the suggestion by reviewer #1.The data provided in main Fig. 6 demonstrate that the cytokine release requires the presence of residual vector and the CAR.Reporter gene encoding LVs or empty LVs do not induce cytokine release.Monocytes enhance cytokine release.
2. The authors show that CAR T are twice as prevalent in the bone marrow of non-tumorbearing mice compared to stCAR T cells.How does their expression compare in the presence of Nalm6 tumor in NSG-SGM3 mice?This would be more relevant to treatment of cancerbearing patients.
The requested data are now provided in Fig. EV3A.
3. How does the murine innate cell compartment (as shown in Fig. 2F) differ between Nalm6bearing mice treated with CAR T versus stCAR T?
In Fig. EV3B we do now show quantifications of the murine immune cell populations in tumor bearing mice compared to tumor-free mice ("wo target").There were increased levels of immune cells especially macrophages detected.We are now mentioning this on line 191-195.
Minor revisions: 1. Fig. 1C: Almost 80% of CAR T cells are double negative (DN)--is this data correct?I would expect more CAR+ by this point in culture.
The data are correct.This is a very early time point compared to a 10-21 days expansion for clinical applications.As you can see in Fig. 1E the amount of CAR positive cells is increasing over the next few days to around 60%, which is well in range of CART cells manufactured for clinical applications.
2. Labeling is off in the text for Fig. 1E-I.
Thank you for making us aware of this error.We have corrected the wrong figure references.
3. Data is not presented in order--i.e.some of Suppl.Fig. 3 is discussed in the text prior to jumping back to Supp.Fig. 2.This made it difficult to follow and would flow better if the data was introduced in the correct order.
We have corrected this.
4. In Supp.Fig. 8, changing the axis of the middle plot (CAR T) to a max of 8 to match the other two graphs will make your data more striking.As it's currently graphed, it appears that the severity of CRS is comparable between stCAR T and CAR T.
Thank you, we have revised as suggested (now Fig. EV5B).
5. Interferon gamma is written as both IFN-g and INF-g in the manuscript--please choose one and make consistent.
We have corrected this all over.
16th Feb 2024 1st Revision -Editorial Decision 16th Feb 2024 Dear Prof. Buchholz, Thank you for submitting your revised manuscript.We have now received the report from the referees who re-reviewed your manuscript.As you will see below, they are satisfied with the revisions, and I will therefore be able to accept your manuscript once the following editorial points will be addressed: 1/ Manuscript text: -Please accept the previous changes, and only keep in track changes mode any new modification.
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5/ I introduced minor modifications in your synopsis text, please let me know if you agree or amend as you see fit:
To make CAR T cells available to all patients, various strategies facilitating the manufacturing process are followed.Short-term (st) CAR T cells are administered shortly after exposure to lentiviral vector (LV) particles.Here, their preclinical safety was assessed ex vivo and in vivo.
• stCAR T cells contain residual vector components on their surface, in particular the vesicular stomatitis virus (VSV) glycoprotein • NSG-SGM3 mice develop severe CRS-like symptoms rapidly after stCAR T cell administration • Release of CRS-typical cytokines occurs in absence of tumor cells and is enhanced by monocytes • The interplay between CAR and LV proteins is the main trigger for cytokine release 6/ As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329),EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts.This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript.Let us know whether you agree with the publication of the RPF.Please note that the Authors checklist will be published at the end of the RPF.This is a very interesting paper exploring the murine models for assessing CRS in CAR T treatment and their data provide important clues.However, the controls used in the experiments were not ideal.This paper could improve significantly if this caveat is solved.
Referee #1 (Remarks for Author): All the questions are well addressed and I recommend publishing this paper.

Referee #3 (Remarks for Author):
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(Reagents and ToolsTable, Materials and Methods, Figures, Data Availability Section) Provide species, strain, sex, age, genetic modification status.Provide accession number in repository OR supplier name, catalog number, clone number, OR RRID.

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(Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section)This checklist is adapted from Materials Design Analysis Reporting (MDAR) Checklist for Authors.MDAR establishes a minimum set of requirements in transparent reporting in the life sciences (see Statement of Task: 10.31222/osf.io/9sm4x).Please follow the journal's guidelines in preparing your the data were obtained and processed according to the field's best practice and are presented to reflect the results of the experiments in an accurate and unbiased manner.

Checklist for Life Science Articles (updated January ideally
, figure panels should include only measurements that are directly comparable to each other and obtained with the same assay.plots include clearly labeled error bars for independent experiments and sample sizes.Unless justified, error bars should not be shown for technical the exact sample size (n) for each experimental group/condition, given as a number, not a range; a description of the sample collection allowing the reader to understand whether the samples represent technical or biological replicates (including how many animals, litters, cultures, etc.

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Include a statement confirming that informed consent was obtained from all subjects and that the experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.State if relevant permits obtained, provide details of authority approving study; if none were required, explain why.
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and III randomized controlled trials
Table, Materials and Methods, Figures, Data Availability Section) State if relevant guidelines or checklists (e.g., ICMJE, MIBBI, ARRIVE, PRISMA) have been followed or provided.Not Applicable For tumor marker prognostic studies, we recommend that you follow the REMARK reporting guidelines (see link list at top right).See author guidelines, under 'Reporting Guidelines'.Please confirm you have followed these guidelines., please refer to the CONSORT flow diagram (see link list at top right) and submit the CONSORT checklist (see link list at top right) with your submission.See author guidelines, under 'Reporting Guidelines'.Please confirm you have submitted this list.Reagents and Tools Table, Materials and Methods, Figures, Data Availability Section) (